The present invention generally relates to methods for treating anemia. More specifically, the present invention relates to methods for treating sickle cell anemia using protected forms(s) of organic aldehydes.
Sickle cell disease is a hemolytic disorder, which affects, in its most severe form, approximately 80,000 patients in the United States (see, for example, D. L. Rucknagel, in R. D. Levere, Ed., Sickle Cell Anemia and Other Hemoglobinopathies, Academic Press, New York, 1975, p.1). The disease is caused by a single mutation in the hemoglobin molecule; xcex26 glutamic acid in normal adult hemoglobin A is changed to valine in sickle hemoglobin S. (see, for example, V. M. Ingram in Nature, 178:792-794 (1956)). Hemoglobin S has a markedly decreased solubility in the deoxygenated state when compared to that of hemoglobin A. Therefore, upon deoxygenation, hemoglobin S molecules within the erythrocyte tend to aggregate and form helical fibers that cause the red cell to assume a variety of irregular shapes, most commonly in the sickled form. After repeated cycles of oxygenation and deoxygenation, the sickle cell in the circulation becomes rigid and no longer can squeeze through the small capillaries in tissues, resulting in delivery of insufficient oxygen and nutrients to the organ, which eventually leads to local tissue necrosis. The prolonged blockage of microvascular circulation and the subsequent induction of tissue necrosis lead to various symptoms of sickle cell anemia, including painful crises of vaso-occlusion.
Now, most patients with sickle cell disease can be expected to survive into adulthood, but still face a lifetime of crises and complications, including chronic hemolytic anemia, vaso-occlusive crises and pain, and the side effects of therapy. Currently, most common therapeutic interventions include blood transfusions, opioid and hydroxyurea therapies (see, for example, S. K. Ballas in Cleveland Clin. J. Med., 66:48-58 (1999). However, all of these therapies are associated with some undesirable side-effects. For example, repeated blood transfusions are known to be associated with the risks of transmission of infectious disease, iron overload, and allergic and febrile reactions. Complications of opioid therapy may include addiction, seizures, dependency, respiratory depression and constipation.
Hydroxyurea, an inhibitor of ribonucleotide reductase, acts by impairing DNA synthesis in cells (see, for example, J. W., Yarbro in Semin. Oncol., 19:1-10 (1992). For decades, hydroxyurea has been used clinically as an anti-cancer agent for the treatment of leukemia, skin and other cancers. Since early 1980, hydroxyurea has been used to treat patients with sickle cell disease. Sickle cell patients treated with hydroxyurea often seem to have fewer painful crises of vaso-occlusion, fewer hospitalizations and fewer episodes of acute chest syndrome (See, for example, S. Charache et al. in New Engl. J. Med., 332:1317-1322 (1995); S. Charache et al. in Med., 75:300-326 (1996); and J. L. Bauman et al. in Arch. Intern Med., 141:260-261 (1981)). It appears that hydroxyurea treatment increases fetal hemoglobin levels in the red cell, which in turn inhibits the aggregation of sickle cell hemoglobin. However, not all patients in these studies benefited from hydroxyurea treatment, and painful crises of vaso-occlusion were not eliminated in most patients. In fact, a recent clinical trial showed that after a 2-year treatment, fetal hemoglobin levels of patients assigned to the hydroxyurea arm of the study did not differ markedly from their pretreatment levels (see, for example, S. Charache in Seminars in Hematol., 34:15-21 (1997)). Thus, the mechanism of action of hydroxyurea in the treatment of sickle cell anemia remains unclear.
In addition to the limited effectiveness of hydroxyurea therapy, such treatment causes a wide range of undesirable side-effects. The primary side-effect of hydroxyurea is myelosuppression (neutropenia and thrombocytopenia), placing patients at risks for infection and bleeding. In addition, long-term treatment with hydroxyurea may cause a wide spectrum of diseases and conditions, including multiple skin tumors and ulcerations, fever, hepatitis, hyperpigmentation, scaling, partial alopecia, atrophy of the skin and subcutaneous tissues, nail changes and acute interstitial lung disease (see, for example, P. J. M. Best et al. in Mayo Clin. Proc., 73:961-963 (1998); M. S. Kavuru et al. in Cerebral Arterial Thrombosis, 87:767-769 (1994); M. J. F. Starmans-Kool et al. in Ann. Hematol., 70:279-280 (1995); and M. Papi et al. in Am Acad. Dermatol., 28:485-486 (1993)).
Since sickle cell disease is a genetic disease, in theory, the gene therapy approach should be considered. In fact, gene therapies employing either ribozyme-mediated or retroviral vector-mediated approaches to replacing the defective human xcex2-globin gene are being actively developed for the treatment of sickle cell disease (see, for example, D. J. Weatherall, Curr. Biol., 8:R696-8 (1998); and R. Pawliuk et al., Ann. N.Y. Acad. Sci., 850:151-162 (1998)). However, the gene therapy approach to treating sickle cell disease involves bone marrow transplantation, a procedure which has its own inherent toxicities and risks (for a review, see, C. A. Hillery in Curr. Opin. Hematol., 5:151-5 (1998)). Thus, there is still a need to develop new and more effective therapeutic agents against sickle cell disease.
The solution behavior of hemoglobin S can be modified chemically, particularly to change its low oxygen affinity and tendency to aggregate upon deoxygenation. Among various covalent modifications, blocking of amino groups of hemoglobin, which can be accomplished under mild conditions, seems to be most favorable and pharmaceutically acceptable. For instance, vanillin (4-hydroxy-3-methoxybenzaldehyde) and other related aromatic aldehydes under physiological conditions are known to bind to the free amino groups of hemoglobin S via the classic Schiff base formation as follows (see, for example, R. H. Zaugg et al. in J. Biol. Chem., 252:8542-8548 (1977)):
Hbxe2x80x94NH2+Rxe2x80x94CHO⇄Hbxe2x80x94Nxe2x95x90CHxe2x80x94R+H2O.
Vanillin is a flavorant present in foods and beverages and has been granted GRAS (generally regarded as safe) compound status by the FDA. No toxicity was observed when vanillin was given to rats at high levels for extensive periods (see, for example, E. C. Hagan et al. in Food Cosmet. Toxicol., 5:141 (1967)). For example, no significant differences were observed between test and control rats with respect to body and organ weights, hemotology, and histopathology when rats received vanillin at 1.0% of the diet for 16 weeks, 2.0% and 5.0% for 1 year, or 0.5%, 1.0% and 2.0% for 2 years.
Schiff base formation between hemoglobin S and vanillin produced a marked increase in oxygen affinity and shifts the oxy⇄deoxy equilibrium in favor of the oxy form for hemoglobin S, both in solution and in intact red cells. The locations where vanillin binds to hemoglobin S have also been characterized by X-ray crystallography (see, for example, D. J. Abraham et al. in Am. J. Heinatol., 77:1334-1341 (1991)). Vanillin was shown to bind near His 103xcex1, Cys 104xcex1 and Gln 131xcex2, with a secondary binding site located between His 116xcex2 and His 117xcex2, a site that has been implicated as a polymer contact residue. Ektacytometric studies also demonstrated that vanillin can inhibit the polymerization process of hemoglobin S under deoxygenated conditions (see, for example, Abraham et al. supra). Together, these studies indicate that vanillin may exhibit two related mechanisms of action as a potential antisickling agent. It not only inhibits sickle polymerization formation, but also shifts the hemoglobin-oxygen association curve to the left, with an increase in the solubility of hemoglobin S molecules. Both mechanisms could lead to the reduction of vaso-occlusion episodes.
Hemoglobin concentration within red cells is about 5 mmoles/liter. Assuming a blood volume of 4 liters in a 60-kg person, about 1 gram of vanillin would be needed in the circulation to exert its anti-sickling effects, taking into account the accumulative increases in the amount of vanillin-HbS adduct. However, orally administered vanillin is poorly bioavailable because of its extensive metabolism in the intestines and liver (see, for example, L. P. Strand and R. R. Scheline in Xenobiotica, 5:49-63 (1975)).
Accordingly, there is still a need in the art for new methods for treating sickle cell anemia. In addition, there is a need for combination treatments that utilize compounds useful in treatment of sickle cell anemia in conjunction with other drugs that are useful in treating sickle cell anemia or one or more of the symptoms associated with sickle cell disease.
The present invention overcomes many of the problems in the art by providing methods for treatment of sickle cell anemia and methods for combination treatment of sickle cell anemia and one or more of the manifestations of sickle cell disease. The invention is based upon the discovery that certain protected forms of organic aldehydes have utility in treatment of sickle cell anemia, for example, by providing a source of organic aldehyde that forms a Schiff base adduct with hemoglobin S in whole blood.
Accordingly, in accordance with the present invention there are provided methods for treatment of sickle cell anemia in a subject in need thereof comprising administering to the subject an effective amount of a protected organic aldehyde, such as, for example, a thiazolidine having the chemical structure I, 
wherein R1 is xe2x80x94OH, alkoxy, substituted alkoxy, cycloalkoxy, substituted cycloalkoxy, aryloxy, substituted aryloxy, heteroaryloxy, substituted heteroaryloxy, or N(R5)2 wherein each R5 is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, and the like,
wherein R2 is H or xe2x80x94Xxe2x80x94R6 wherein X is carbonyl or sulfonyl and R6 is alkyl, substituted alkyl, aryl, substituted aryl, alkylaryl, or substituted alkylaryl, and the like, and
wherein one of R3 and R4 is H and the other is H, alkyl substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl, and the like.
Particularly useful thiazolidines contemplated for use in practice of the present invention are those wherein R3 and R4 are derived from an arylaldehyde, such as those having the structure II 
wherein each of R7, R8, and R9 are optional and, if present, are independently xe2x80x94OH, alkyl, substituted alkyl, alkoxy, cycloalkoxy, acyloxy, cycloacyloxy, F, Cl, Br, NO2, cyano, or the like.
In accordance with another embodiment of the present invention, there are provided methods for increasing in vivo stability of therapeutic organic aldehyde(s). In the invention in vivo stabilization method, the aldehyde is converted into a protected form thereof, e.g., the aldehyde may be incorporated into a thiazolidine having the chemical structure I above. The oral availability of the organic aldehyde in such invention compounds is increased by from about 4 to about 10 times compared to that of the free organic aldehyde, making them suitable for oral administration. Hence, treatment of sickle cell anemia according to the invention methods affords the considerable advantage that the therapeutic compounds can be administered orally to a subject in need thereof, thereby avoiding the discomfort and inconvenience to the subject of injections.